https://www.facebook.com/events/486473718220320/​The Endocannabinoid System: Truths, Half-Truths, and UnknownsScientists frequently speak in caveats because there are few absolute truths in science; what is "true" (relevant) in one set of circumstance may not be true at all in a different context.

The endocannabinoid system mediates a range of physiological responses that seem paradoxical. Activation of the CB2 receptor by THC, for example, suppresses immune cells that cause Crohn's Disease, but does not cause an overall state of immune-compromise. Cannabidiol (CBD), despite its structural similarity to THC, is not psychoactive, does not bind either of the two known endocannabinoid receptors (CB1 and CB2), yet somehow mimics THC's anti-inflammatory properties.

This month's Frank Talk will explore some of what is currently known about the endocannabinoid system, attempt to dispel a few misunderstandings, and discuss promising new avenues of research.

Thrips species feed on a wide array of plants and other insects by first puncturing them with their mandibles (clearly visible in image), then drawing out the nutrients through a tube-like structure in the mandibles called a stylet. Some species of thrips are predatory, and may be deliberately released in cannabis grows to combat spider mites and other parasites, but the majority of thrips are phytophagous ("plant-eating") insects that feed on chloroplasts from epidermal and mesophyll cells on the surface of the plant.

Wikipedia article for general information on thrips:https://en.wikipedia.org/wiki/Thrips

The Chemicals Between Us: ImidaclopridPart 1 of a series examining the implications of pesticide contamination in cannabis products.

This week's Frank Talk will compare EPA-established tolerance levels of imidacloprid in other commodities versus levels found in cannabis flower and concentrates, imidacloprid's mechanism of action, and discussion of potential health concerns for cannabis consumers.

24-March-2016 Update: The infection may have been caused by not one, but two fungal organisms; an epiphytic form of powdery mildew (possibly Podosphaera macularis) and endophytic false powdery mildew (probably Trichothecium roseum). More details and full update later.

Powdery Mildew Disease of CannabisColorado Green Lab, in collaboration with Hempirical, is examining the life cycle, pathogenicity, and infection patterns in PM-resistant and PM-susceptible strains of cannabis.

Powdery mildew may cause substantial economic loss directly by negatively impacting plant growth and flower quality, and indirectly if treated plants are rendered unsuitable for human consumption by accumulation of unsafe levels of fungicide residue. This is particular relevant where microorganisms have developed high levels of resistance to common fungicides (Demethylation Inhibitors such as myclobutanil), and cultivators apply ever-increasing amounts of fungicide just to keep the mildew in check. By elucidating the underlying cellular and genetic mechanisms behind cannabis powdery mildew susceptibility (and resistance), we aim to develop more effective means of combating the pathogen and reduce cultivation losses.

Background Powdery mildew is not caused by a single organism, but by many different species of fungi. Most PM-species are highly specific to their preferred host and have evolved to attack a limited range of somewhat similar plants (e.g. cucumber mildew fungi cannot cause oak tree mildew, but can cause disease in squash plants). All PM-species are biotrophic, meaning they require living host tissue to grow and reproduce, and most share common infection mechanisms, with similar life cycles.

Powdery mildew fungi are predominantly epiphytic ("epi" = "upon, near to", "phyt" = "plant") and grow on and around the surface of the plant, with some variants that also produce full or partially endophytic ("endo" = "within") mycelium and hyphae inside the infected plant tissue.

Infection of Susceptible Afghan IndicaPowdery mildew infection of cannabis occurs on the adaxial (upper) surface of the leaf, which is coated with fine hairs (A-D) that may facilitate spore adhesion and subsequent infection.

Infection occurs in stages. Spores landing on the upper surface of the leaf (E) germinate and form appressorium (F), flattened appendages that facilitate penetration.

Appressorium form one or more haustorium (G), which are specialized appendages that punch a hole in the leaf surface, then act as feeding tubes that withdraw nutrients from the living plant.

In PM-resistant cannabis strains, stomata automatically close soon after spore attachment (not shown), possibly to shut down photosynthesis by the affected tissue and starve out the infecting fungus. In susceptible cannabis strains (e.g. "Blueberry"), stomata are locked open directly below the site of the infection (H), allowing the infected tissue to continue photosynthesis and direct nutrients to the invading fungal haustorium.

Following successful penetration of the leaf surface, filamentous mycelial growth invades the plant tissue, spreading throughout the interior of the leaf (I) and emerging from the lower (abaxial) leaf surfaces through stomata (J). Mycelium also forms on the exterior of the upper leaf surface and forms spore-bearing conidiophores (K), which release spores into the air and continue the cycle of infection.

Surprise GuestSay hello to my little friend! Russet mite (probably...I'm not an entomologist) was found unexpectedly on the surface of a PM-infected leaf I was examining.

Microscopy NotesLeaves were destained with Visikol (http://visikol.com/) to improve visualization. For added contrast of fungal and plant structures, some samples (those with reddish hue) were stained with modified Lugol's reagent, followed by dilute sulphuric acid. Thanks and credit to Koroch et al for the useful staining technique described in the literature referenced below.

Powdery mildew organisms are highly specific for their host, and P. fraxini, the causal agent shown here, doesn't infect cannabis. We used lilac leaves and associated PM as control samples to validate some new sample preparation, staining and microscopy techniques.

Powdery mildew genera are grouped into five tribes:Phyllactineae, Erysipheae, Blumeriae, Golovinomyceteae, and Cystotheceae.

The tribes can be identified by several criteria, including the type of host plant (Ex. Phyllactineae infects trees and shrubs), and by microscopic examination of their chasmothecium and appendage morphology, as well as shape and structure of spores.

Cindy and I collected took Pee Em from a highly infected Lilac bush and brought it back to the lab. The dirty little %$#@ fungus wouldn't talk tho, and that's when things got a bit rough. We made the parasite spill its guts, all of them.

Host plant (Lilac) and appendage morphology ("spines") positively identifies genera as Phyllactinia. Organism is most probably P. fraxini, which is known to infect Lilac leaves.

Overview On March 23rd, several Denver-based marijuana grow operations were ordered to quarantine plants after it was determined they were treated with Eagle 20, a fungicidal pesticide (1). Myclobutanil-based fungicides, including Eagle 20, are applied to a wide range of edible agricultural products (grapes, apples, spinach, etc). When applied correctly, myclobutanil is known to have low toxicity to humans. Myclobutanil-based fungicides, including Eagle 20EW, are not currently approved for use in the United States on tobacco, the only (other than marijuana) smokable agricultural commodity. The toxicity and health effects of myclobutanil in the context of combustion/inhalation (versus ingestion) have not been assessed. The following analysis summarizes some of the known chemical and physical properties of myclobutanil, and highlights the potential health implications of using this chemical on marijuana.Mode of Action Myclobutanil is the active ingredient in several brands of pesticides, including Eagle 20EW. Myclobutanil works by blocking a key enzyme involved in fungal cell membrane synthesis, leading to abnormal cell growth and eventual death of the fungal pathogen (2) Myclobutanil is a systemic fungicide, meaning it is absorbed at the site of application (ex. leaf) and distributed throughout the rest of the plant, thereby providing more comprehensive protection from fungal infection (2). As a systemic chemical, myclobutanil cannot be removed by washing treated crops, although residue will decrease in plant tissues over time. The final remaining residue levels vary considerably and are highly dependent on the rate of application, the time of last application before harvest, and how well the specific plant clears the chemical from its system.

Myclobutanil Tolerance Levels The Environmental Protection Agency is responsible for regulating the pesticides used by growers to protect crops and for setting limits on the amount of pesticides that may remain in or on foods marketed in the USA. These limits on pesticides left on foods are called "tolerances" in the U.S (3).The EPA establishes tolerances or each pesticide based on the potential risks to human health posed by that pesticide, using actual or estimated residue data, as well non-human toxicity studies, to reflect real-world exposure as closely as possible (3). Tolerance levels for myclobutanil were established for exposure via inhalation, absorption through the skin during pesticide application to crops, and ingestion of agricultural commodities treated with myclobutanil. Myclobutanil absorbed by the most common route, dietary exposure, is metabolized by gut enzymes and the liver prior to entering the bloodstream (4,5). Myclobutanil absorbed via inhalation enters the bloodstream directly via the lungs. I. The human health effects from the combustion and inhalation of myclobutanil have not been evaluated Tolerance levels and toxicity studies for myclobutanil on edible products should not be used for evaluating the safety of myclobutanil on marijuana. Passage of pesticides into the bloodstream varies considerably between inhalation and ingestion routes of exposure, and the application of high temperature is known to alter the chemical composition of myclobutanil. Unfortunately, very little information is available to evaluate myclobutanil in the context of tobacco use, as Eagle 20 and myclobutanil-based fungicides are not approved for use on tobacco plants in the United States (6,7). Myclobutanil is approved for use on tobacco cultivated in China, however, and a 2012 study has demonstrated that 10% or more of the active pesticide remains on tobacco leaves up to 21 days after treatment, with residue present from 0.85 parts per million (ppm) up to 3.27 ppm (8). Using tobacco as a model for pesticide retention, it is probable a considerable amount of myclobutanil may remain present in cannabis weeks after fungicide application.

As shown in Figure 1 above, myclobutanil decomposes, its triazole (Figure 1, blue), cyanide (Figure 1, red) and chlorine (Figure 1, green) moieties are released and form toxic gases, including hydrogen cyanide (HCN) and hydrochloric gas (HCl). Of the three primary decomposition products formed, HCN holds the greatest concern. Chronic exposure to dilute hydrogen cyanide (ex. 0.008 parts per million) is not immediately deadly (10), but is known to cause serious neurological, respiratory, cardiovascular, and thyroid problems (11, 12, 13). Cannabis retaining even marginal amounts of myclobutanil (ex. 0.03 ppm) could potentially expose consumers to non-lethal, but clinically relevant levels of HCN. III. Myclobutanil is co-extracted with cannabinoids during concentrate production Studies of two other conazole fungicides, tebuconazole and propiconazole, have demonstrated that these chemicals are co-extracted during production of essential oils (14). Moreover, the process of extraction, treatment, and concentration can cause tebuconazole and propiconazole pesticide residue to accumulate at levels 250 times higher than the starting material (14). Myclobutanil is highly soluble in many of the solvents used in cannabinoid extraction (ex. ethanol, butane, and carbon dioxide)(15,16), and unquestionably co-extracts with cannabinoids during concentrate production. The process of removing residual solvent and increasing cannabinoid concentration very likely increases levels of myclobutanil, and other chemically-similar pesticides.ConclusionThe Colorado Department of Agriculture has identified and published a list of "minimum risk pesticides" that pose little or no risk to human health (18) and are allowable for use on marijuana during cultivation. Eagle 20 (myclobutanil) is not on this list, but the absence of regulatory oversight has contributed to its widespread use in marijuana cultivation in Colorado.